Team:EPFL/Demonstrate

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Building a cell-free biosensor for protein detection based on aptamers for target recognition and toeholds for signal generation




How does our biosensor work



Figure 1: Scheme of our biosensor concept




Our system relays on three major building blocks:

  • Aptamer pair detects the presence of a protein in a sample
  • Aptamer can trigger the toehold
  • Translation of the downstream reporter for signal generation in our home-made cell-free lysate



    • 1. How do we detect the presence of a target protein

    Figure 2: Protein detection scheme using ELISA derived sandwich-based assay


    To demonstrate the ability of our aptamer pair to bind to their target protein Thrombin we used microfluidics, and measured the fluorescence of the Cy3 probe which is attached to the second aptamer. If high levels of fluorescence are measured, this indicates that Thrombin was bound between the two aptamers.

    Our experimental set up was as follows: the biotinylated Aptamer 1 was flown first, sticking to the surface of the chip. Then, Thrombin (target protein) was flown only in the top half of the chip and finally the fluorescently labeled Aptamer 2 with trigger extension in the top and bottom half of the chip (see Figure 3a for schematics).

    Figure 3a & b: Sandwich immunoassay with Thrombin Aptamers 1 and 2 trigger extension in buffer
    Right side : Fluorescent read-out




    2. How do we generate a colorometric signal upon protein detection


    To generate the signal upon the human Thrombin detection, we used toehold switches and the aptamer trigger that recognizes the human Thrombin.

    Figure 4: Toehold opens up after aptamer trigger extension annealing, beta-galactosidase is expressed

    In the graph below, we should the titration of the aptamer trigger we performed which show that a the toehold is trigger by this DNA at different concentrations

    Figure 5: Aptamer trigger titration in cell lysate

    To see how the levels of absorbance translate to reactions in tubes, we prepared tube reactions of the toehold with and without the aptamer trigger extension to assess whether we could see the color change by eye.
    Figure 6a: Aptamer trigger with toehold in lysate reaction incubated for 2 hours at 37°C
    Figure 6b: Toehold without aptamer trigger in lysate reaction incubated for 2 hours at 37°C


    3. Streamline toehold design by writing a software

    Figure7 :Toehold Designer logo

    Generating new toehold sensors requires in-silico processing. It is a quick step (~5 min) if a tool that pipelines the required processes is available. In the scope of our project we generated our own switches targeting Hepatitis C viral RNA and sucessfully proven that the toeholds are functional, i.e they unfolded only in the prescence of a complementary sequence to allow the translation of the downstream reporter lacZ.

    Figure 8: Toehold A in the iGEM backbone psB1C3

    We tested the ToeholdDesigner's output, and chosen the best 4 toeholds for a unique sequence of the Hepatitis C virus we have found using different online resources such as BLAST, in our home-made lysates.


    As shown below, we demonstrate that we can design functional toeholds using ToeholdDesigner
    Figure 9a: Kinetic measurements of the 4 generated toeholds in a usual lysate reaction
    Shaded error graphs
    Figure 9b: End point measurements of the 4 generated toeholds in a usual lysate reaction